1. Field of the Invention
The present invention relates generally to the manipulation of genetic materials and particularly to the manufacture and use of specific DNA sequences useful in recombinant procedures to secure the production of peptides having one or more properties of nucleotide pyrophosphohydrolase.
2. Background of the Art
Calcium pyrophosphate dihydrate (CPPD) crystal deposition in human articular cartilage is a common age-dependent event often associated with degeneration of articular tissues and/or with acute attacks of arthritis (D. McCarty, et al., 56 Ann. Intern. Med. 711-737 (1962)). Prospects for preventing or reversing CPPD crystal deposition depends on a better understanding of inorganic pyrophosphate (PP.sub.i) metabolism in joint tissues. Synovial fluid PP.sub.i levels are nearly always greater than those of plasma and correlate directly with the degree of radiographically evident joint degeneration (L. Ryan, et al., In Arthritis and Allied Conditions, 12th ed., D. McCarty, et al., editors, pp. 1835-1855 (1993)). PP.sub.i is released into the ambient medium when cartilage slices or isolated chondrocytes but not other joint tissues are cultured in vitro, suggesting that this tissue is the source of synovial fluid PP.sub.i. Detergent extracts of CPPD crystal-encrusted degenerated cartilage contained increased nucleotide pyrophosphohydrolase (NTPPHase) activity relative to normal or degenerated cartilage without crystals (J. Tenenbaum, et al., 24 Arthritis Rheum. 294-300 (1981)). This enzyme generates PP.sub.i from ATP and other nucleotides. NTPPHase is an ectoenzyme in canine (L. Ryan, et al., 27 Arthritis Rheum. 404-409 (1984)) and in human (A. Caswell, et al., 847 Biochem. Biophys. Acta. 40-47 (1985)) chondrocytes. ATP levels were significantly higher in knee joint fluids containing CPPD crystals compared with fluids from osteoarthritis (217 vs. 98 nM) (L. Ryan, et al., 18 J. Rheumatol. 716-720 (1991)) despite higher activity of NTPPHase in the former (J. Rachow, et al., 28 Arthritis Rheum. 1283-1288 (1985)).
EctoNTPPHase activity is also associated with vesicles derived from chondrocytes in epiphyseal cartilage, termed matrix vesicles, and is thought by some investigators important for in vitro calcification (S. Siegal, et al., 258 J. Biol. Chem. 8601-8607 (1983); A. Caswell, et al., 924 Biochem. Biophys. Acta. 276-283 (1987)). Addition of ATP to vesicles isolated from adult porcine (B. Derfus, et al., 35 Arthritis Rheum. 231-240 (1992)) or human (L. Ryan, et al., 35 Arthritis Rheum. 1520-1525 (1992)) hyaline articular cartilage, termed articular cartilage vesicles (ACV), formed a mineral phase tentatively identified as monoclinic CPPD.
The inventors have noted an enzyme activity on the exterior of many cells which generates inorganic pyrophosphate (PP.sub.i), a necessary constituent of pathologic crystals which form in cartilages of patients with a specific form of arthritis termed calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. The enzyme is called nucleotide pyrophosphohydrolase (NTPPH) because of one reaction which it catalyzes, the hydrolysis of nucleoside triphosphates such as ATP to a nucleoside monophosphate and PP.sub.i. This enzyme activity is very likely an important if not essential factor in the generation of PP.sub.i which then forms CPPD crystals.
There are many forms of NTPPH. One specific form has been characterized in our laboratory and is termed 127 kD NTPPH, based upon its molecular weight. Antibodies raised against porcine (pig) 127 kD NTPPH have been used to determine the tissue distribution, and 127 kD NTPPH was found only in tissues in or around joints. Within these articular tissues, 127 kD is most concentrated in cartilage. In chondrocytes, the cells of cartilage, 127 kD NTPPH is restricted to vesicles termed articular cartilage vesicles (ACV). These are important structures which can mineralize to form CPPD crystals when a substrate such as ATP is added. These are the only structures that have ever been found to serve as a nidus for CPPD formation and they are very enriched in the 127 kD enzyme.
The native 127 kD enzyme is attached to the membranes of these vesicles. However, antibody to 127 kD NTPPH also identifies putative breakdown fragments that are of lower molecular weight, that are soluble, and that still possess enzyme activity. These have been identified in the joint fluid surrounding cartilage and in the serum of humans, but not in tissues. In joint fluid and serum the activity of this enzyme has been found to be elevated in patients who have CPPD crystals in their cartilage and to a lesser degree in patients who have osteoarthritis.
It would be advantageous to clone cDNA encoding nucleotide pyrophosphohydrolase so that sufficient quantities of NTPPH can be generated for use in further studies and diagnostic testing.